Over the last several years, a variety of approaches to the synthetic or semi-synthetic preparation of insulin have been advanced. Insulin is a molecule having two peptide chains, an A-chain containing 21 amino acid residues and a B-chain containing 30 amino acid residues. These chains contain three disulfide bridges, each formed from two cysteinyl residues. Two of the disulfide bridges join the A-chain to the B-chain. The bridges are formed from the cysteinyl residues at A-6 and A-11, A-7 and B-7, and A-20 and B-19, respectively.
One general method for insulin production is via proinsulin or a proinsulin-like molecule. Proinsulin is a single chain polypeptide in which the N-terminus of the insulin A-chain is linked through a connecting peptide with the C-terminus of the insulin B-chain, the appropriate cysteine residues being joined by disulfide bonds. Human proinsulin, e.g., has 86 amino acid residues, 35 of which make up the connecting peptide. Yanaihara et al., Diabetes 27 (Suppl. 1) 149-160 (1978) describe the synthesis of a variety of connecting peptides and human proinsulin.
Other proinsulin-like molecules have been described in the literature, the principal differences from proinsulin being the structure of the moiety which connects the insulin A- and B-chains and the point at which such connection is made.
Thus, Busse et al., Biochemistry 15, No. 8, 1649-1657 (1976) report a linkage comprising two methionyl residues joined at their N-terminus by a carbonyl group and the resulting moiety joined to the N.sup..alpha. -terminus of the A-1 glycyl and the N.sup..epsilon. -terminus of the B-29 lysyl.
Similarly, other connecting moieties have been described. See, for example, Geiger et al., Biochem. and Biophys. Res. Comm. 55, 60-66 (1973); Brandenburg et al., Hoppe-Seyler's Z. Physiol. Chem. bd. 354, 613-627 (1973); U.S. Pat. Nos. 3,847,893; 3,907,763; 3,883,496; 3,883,500; and 3,884,897.
In any of these approaches for production of insulin via a single chain comprising insulin A- and B-chains joined through a defined moiety, direct interconnection of the insulin A- and B-chains must be carried out by formation of three disulfide bridges from the six cysteinyl residues present on the A- and B-chains. Following disulfide bond formation, the original connecting moiety is removed with formation of insulin.
In effecting this approach to insulin production, an efficient and ready method for correct disulfide bridge formation is highly desirable. In general, the literature methods for forming the disulfide bridges involve air oxidation of the corresponding --SH structures. Furthermore, since it is recognized that the --SH structure is unstable, the precursor normally is generated with an S-protecting group, typically an S-sulfonate (--S--SO.sub.3.sup.-) moiety. Thus, the literature methods involve a two-step sequence, i.e., reduction of the S-sulfonate to --SH by treatment with a mercaptan followed by air oxidation of the formed --SH compound.
It now has been discovered that a facile and highly efficient method for direct conversion of the S-sulfonate to the desired disulfide insulin precursor is available. The process does not contemplate a reduction-oxidation sequence. Instead, a direct interchange is effected under conditions that, although not essential, prefer the absence of an oxidizing agent. It is to such a process that this invention is directed.
One possible exception in the prior art to the general two-step method, applied, however, to combination of insulin A- and B-chains and not to disulfide formation from a linear chain S-sulfonate insulin precursor, is represented by Dixon et al., Nature 188, 721-724 (1960), which perhaps implies production of insulin by combination of A- and B-chain S-sulfonates in a single solution. The details of this prior art method are quite sketchy, and the yield, based only on activity of the product recovered, represented only 1-2%. A later publication, Dixon, Proc. Intern. Congr. Endocrinal. 2nd London 1964, 1207-1215 (1965), appears somewhat to clarify the details of this method, suggesting, in Table IV, page 1211, a two-step process involving anaerobic S-sulfonate reduction followed by oxidation to the disulfide.